The immune system of vertebrates (for example, primates, which include humans, apes, monkeys, etc.) consists of a number of organs and cell types which have evolved to: accurately and specifically recognize foreign microorganisms (“antigen”) which invade the vertebrate-host; specifically bind to such foreign microorganisms; and, eliminate/destroy such foreign microorganisms. Lymphocytes, as well as other types of cells, are critical to the immune system. Lymphocytes are produced in the thymus, spleen and bone marrow (adult) and represent about 30% of the total white blood cells present in the circulatory system of humans (adult).
There are two major sub-populations of lymphocytes: T cells and B cells. T cells are responsible for cell mediated immunity, while B cells are responsible for antibody production (humoral immunity). However, T cells and B cells can be considered interdependent—in a typical immune response, T cells are activated when the T cell receptor binds to fragments of an antigen that are bound to major histocompatability complex (“MHC”) glycoproteins on the surface of an antigen presenting cell; such activation causes release of biological mediators (“interleukins”) which, in essence, stimulate B cells to differentiate and produce antibody (“immunoglobulins”) against the antigen.
Each B cell within the host expresses a different antibody on its surface—thus one B cell will express antibody specific for one antigen, while another B cell will express antibody specific for a different antigen. Accordingly, B cells are quite diverse, and this diversity is critical to the immune system. In humans, each B cell can produce an enormous number of antibody molecules (i.e., about 107 to 108). Such antibody production most typically ceases (or substantially decreases) when the foreign antigen has been neutralized. Occasionally, however, proliferation of a particular B cell will continue unabated; such proliferation can result in a cancer referred to as “B cell lymphoma.”
T cells and B cells both comprise cell surface proteins which can be utilized as “markers” for differentiation and identification. One such human B cell marker is the human B lymphocyte-restricted differentiation antigen Bp35, referred to as “CD20.” CD20 is a B-lymphocyte-restricted differentiation antigen that is expressed during early pre-B-cell development and remains until plasma cell differentiation. It is believed by some that the CD20 molecule may regulate a step in the B-cell activation process which is required for cell cycle initiation and differentiation. Moreover CD20 is usually expressed at very high levels on neoplastic (“tumor”) B-cells. The CD20 antigen is appealing for targeted therapy, because it does not shed, modulate, or internalize. Thus, the CD20 surface antigen is an attractive candidate for “targeting” of B cell lymphomas.
In essence, such targeting can be generalized as follows: antibodies specific to the CD20 surface antigen of B cells are, e.g., injected into a patient. These anti-CD20 antibodies specifically bind to the CD20 cell surface antigen of (ostensibly) both normal and malignant B cells; the anti-CD20 antibody bound to the CD20 surface antigen may lead to the destruction and depletion of neoplastic B cells. Additionally, chemical agents or radioactive labels having the potential to destroy the tumor can be conjugated to the anti-CD20 antibody such that the agent is specifically “delivered” to, e.g., the neoplastic B cells. Irrespective of the approach, a primary goal is to destroy the tumor: the specific approach can be determined by the particular anti-CD20 antibody which is utilized and, thus, the available approaches to targeting the CD20 antigen can vary considerably.
For example, attempts at such targeting of CD20 surface antigen have been reported. Murine (mouse) monoclonal antibody 1F5 (an anti-CD20 antibody) was reportedly administered by continuous intravenous infusion to B cell lymphoma patients. Extremely high levels (>2 grams) of 1F5 were reportedly required to deplete circulating tumor cells, and the results were described as being “transient.” Press et al., “Monoclonal Antibody 1F5 (Anti-CD20) Serotherapy of Human B-cell Lymphomas,” Blood 69/2:584–591 (1987).
A potential problem with this approach is that non-human monoclonal antibodies (e.g., murine monoclonal antibodies) typically lack human effector functionality, i.e., they are unable to, inter alia, mediate complement dependent lysis or lyse human target cells through antibody dependent cellular toxicity or Fc-receptor mediated phagocytosis. Furthermore, non-human monoclonal antibodies can be recognized by the human host as foreign proteins; therefore, repeated injections of such foreign antibodies can lead to the induction of immune responses leading to harmful hypersensitivity reactions. For murine-based monoclonal antibodies, this is often referred to as a Human Anti-Mouse Antibody response, or “HAMA” response. Additionally, these “foreign” antibodies can be attacked by the immune system of the host such that they are, in effect, neutralized before they reach their target site.
One approach at compensating for the lack of effector function of murine antibodies is to conjugate such antibodies to a toxin or radiolabel. Lymphocytes and lymphoma cells are inherently sensitive to radiotherapy. Therefore, B cell malignancies are attractive targets for radioimmunotherapy (RIT) for several reasons: the local emission of ionizing radiation of radiolabeled antibodies may kill cells with or without the target antigen (e.g., CD20) in close proximity to antibody bound to the antigen; penetrating radiation, i.e., beta emitters, may obviate the problem of limited access to the antibody in bulky or poly vascularized tumors; and, the total amount of antibody required may be reduced, thereby alleviating the severity of the potential HAMA response. The radionuclide emits radioactive particles which can damage cellular DNA to the point where the cellular repair mechanisms are unable to allow the cell to continue living; therefore, if the target cells are tumors, the radioactive label beneficially kills the tumor cells. Radiolabeled antibodies, by definition, include the use of a radioactive substance which may require the need for precautions for both the patient (i.e., possible bone marrow transplantation) as well as the health care provider (i.e., the need to exercise a high degree of caution when working with radioactivity).
A number of specific antibodies have now been disclosed for which a radioactive label or toxin has been conjugated to the antibody such that the label or toxin is localized at the tumor site. For example, the above-referenced IF5 antibody has been “labeled” with iodine-131 (131I) and was reportedly evaluated for biodistribution in two patients. See Eary, J. F. et al., “Imaging and Treatment of B-Cell Lymphoma” J. Nuc. Med. 31/8:1257–1268 (1990); see also, Press, O. W. et al., “Treatment of Refractory Non-Hodgkin's Lymphoma with Radiolabeled MB-1 (Anti-CD37) Antibody” J. Clin. Onc. 718:1027–1038 (1989) (indication that one patient treated with 131I-labeled IF5 achieved a “partial response”); Goldenberg, D. M. et al., “Targeting, Dosimetry and Radioimmunotherapy of B-Cell Lymphomas with Iodine-131-Labeled LL2 Monoclonal Antibody” J. Clin. Onc. 9/4:548–564 (1991) (three of eight patients receiving multiple injections reported to have developed a HAMA response); Appelbaum, F. R. “Radiolabeled Monoclonal Antibodies in the Treatment of Non-Hodgkin's Lymphoma” Hem./Onc. Clinics of N. A. 5/5:1013–1025 (1991) (review article); Press, O. W. et al “Radiolabeled-Antibody Therapy of B-Cell Lymphoma with Autologous Bone Marrow Support.” New England Journal of Medicine 329/17: 1219–12223 (1993) (iodine-131 labeled anti-CD20 antibody IF5 and B1); and Kaminski, M. G. et al “Radioimmunotherapy of B-Cell Lymphoma with 131I Anti-B1 (Anti-CD20) Antibody”. NEJM 329/7 (1993) (iodine-131 labeled anti-CD20 antibody B1; see also U.S. Pat. No. 5,843,398 to Kaminski). Toxins (i.e. chemotherapeutic agents such as doxorubicin or mitomycin C) have also been conjugated to antibodies. See, for example, PCT published application WO 92/07466 (published May 14, 1992).
U.S. application Ser. Nos. 08/475,813, 08/475,815 and 08/478,967, herein incorporated by reference, disclose radiolabeled therapeutic antibodies for the targeting and destruction of B cell lymphomas and tumor cells. In particular, the Y2B8 antibody is disclosed, which is an anti-human CD20 murine monoclonal antibody, 2B8, attached to yttrium-[90] (90Y) via the bifunctional chelator, MX-DTPA. This radionuclide was selected for therapy for several reasons. The 64 hour half-life of 90Y is long enough to allow antibody accumulation by the tumor and, unlike e.g. 131I, it is a pure beta emitter of high energy with no accompanying gamma irradiation in its decay, with a range of 100 to 1000 cell diameters. The minimal amount of penetrating radiation allows for outpatient administration of 90Y-labeled antibodies. Furthermore, internalization of labeled antibodies is not required for cell killing, and the local emission of ionizing radiation should be lethal for adjacent tumor cells lacking the target antigen.
Patents relating to chelators and chelator conjugates are known in the art. For instance, U.S. Pat. No. 4,831,175 of Gansow is directed to polysubstituted diethylenetriaminepentaacetic acid chelates and protein conjugates containing the same, and methods for their preparation. U.S. Pat. Nos. 5,099,069, 5,246,692, 5,286,850, and 5,124,471 of Gansow also relate to polysubstituted DTPA chelates. These patents are incorporated herein in their entirety.
The specific bifunctional chelator used to facilitate chelation in application Ser. Nos. 08/475,813, 08/475,815 and 08/478,967 was selected as it possesses high affinity for trivalent metals, and provides for increased tumor-to-non-tumor ratios, decreased bone uptake, and greater in vivo retention of radionuclide at target sites, i.e., B-cell lymphoma tumor sites. However, other bifunctional chelators are known in the art and may also be beneficial in tumor therapy.
As also reported in copending application Ser. Nos. 08/475,813, 08/475,815 and 08/478,967 administration of the radiolabeled Y2B8 conjugate, as well as unlabeled chimeric anti-CD20 antibody, resulted in significant tumor reduction in mice harboring a B cell lymphoblastic tumor. Moreover, human clinical trials reported therein showed significant B cell depletion in lymphoma patients infused with chimeric anti-CD20 antibody. In fact, chimeric 2B8 has recently been heralded the nation's first FDA-approved anti-cancer monoclonal antibody under the name of Rituximab® (Rituxan® in the U.S. and Mabthera® in the U.K.).
In addition, U.S. application Ser. No. 08/475,813 discloses sequential administration of Rituxan® with yttrium-labeled Y2B8 murine monoclonal antibody. Although the radiolabeled antibody used in this combined therapy is a murine antibody, initial treatment with chimeric anti-CD20 sufficiently depletes the B cell population such that the HAMA response is decreased, thereby facilitating a combined therapeutic and diagnostic regimen. Moreover, it was shown in U.S. application Ser. No. 08/475,813 that a therapeutically effective dosage of the yttrium-labeled anti-CD20 antibody following administration of Rituxan® is sufficient to (a) clear any remaining peripheral blood B cells not cleared by the chimeric anti-CD20 antibody; (b) begin B cell depletion from lymph nodes; or (c) begin B cell depletion from other tissues.
Thus, conjugation of radiolabels to cancer therapeutic antibodies provides a valuable clinical tool which may be used to enhance or supplement the tumor-killing potential of the chimeric antibody. Given the proven efficacy of an anti-CD20 antibody in the treatment of non-Hodgkin's lymphoma, and the known sensitivity of lymphocytes to radioactivity, it would be highly advantageous for such therapeutic antibodies to become commercially available in kit form whereby they may be readily modified with a radiolabel and administered directly to the patient in the clinical setting.
To this end, U.S. application Ser. No. 09/259,337 discloses methods, reagents and kits for accomplishing radiolabeling of antibodies. Such kits are convenient vehicles for placing these reagents in the clinical setting, in a way that they may be easily produced and administered to the patient before significant decay of the radiolabel or significant destruction of the antibody due to the radiolabel occurs. The kits disclosed in application Ser. No. 09/259,337, herein incorporated by reference, overcome many deficiencies of the prior art which deterred the introduction of such convenient means to commercialize this valuable technology.
The slow introduction of radiolabeling kits to the market may have been due to the poor incorporation efficiencies demonstrated by some known labeling protocols, and the subsequent need to column purify the reagent following the radiolabeling procedure. The delay in development of such kits might also in part be due to the previous lack of accessibility to pure commercial radioisotopes which may be used to generate efficiently labeled products absent subsequent purification. Alternatively, perhaps the reason such kits are generally unavailable is the actual lack of antibodies which have been able to achieve either the approval or the efficacy that Rituxan® has achieved for the treatment of lymphoma in human patients.
For instance, as discussed in U.S. Pat. No. 4,636,380, herein incorporated by reference, it has been generally believed in the scientific community that for a radiopharmaceutical to find clinical utility, it must endure a long and tedious separation and purification process. Indeed, injecting unbound radiolabel into the patient would not be desirable. The need for additional purification steps renders the process of radiolabeling antibodies in the clinical setting an impossibility, particularly for doctors who have neither the equipment nor the time to purify their own therapeutics.
Furthermore, radiolabeled proteins may be inherently unstable, particularly those labeled with radiolytic isotopes such as 90Y, which have the tendency to cause damage to the antibody the longer they are attached to it in close proximity. In turn, such radiolysis causes unreliable efficiency of the therapeutic due to loss of radiolabel and/or reduced binding to the target antigen, and may lead to undesired immune responses directed at denatured protein. Yet without the facilities for labeling and purifying the antibodies on site, clinicians have had no choice but to order therapeutic antibodies already labeled, or have them labeled off site at a related facility and transported in following labeling for administration to the patient. All such manipulations add precious time to the period between labeling and administration, thereby contributing to the instability of the therapeutic, while in effect decreasing the utility of radiolabeling kits in the clinical setting.
Others have claimed to have developed radiolabeling protocols which would be amenable to kit format in that a separate purification step would not be required (Richardson et al. (1987) Optimization and batch production of DTPA-labeled antibody kits for routine use in 111In immunoscintography. Nuc. Med. Commun. 8: 347–356; Chinol and Hnatowich (1987) Generator-produced yttrium-[90] for radioimmunotherapy. J. Nucl. Med. 28(9): 1465–1470). However, such protocols were not able to achieve the level of incorporation that the present inventors have achieved using the protocols disclosed herein, which have resulted in incorporation efficiencies of at least 95%. Such a level of incorporation provides the added benefit of increased safety, in that virtually no unbound label will be injected into the patient as a result of low radioincorporation.
The protocols included in the kits of the invention disclosed in U.S. application Ser. No. 09/259,337 allow rapid labeling which may be affected in approximately a half an hour or as little as five minutes depending on the label. Moreover, as discussed above, the kit protocols disclosed in this application have a labeling efficiency of over 95% thereby foregoing the need for further purification. By foregoing the need for further purification, the half-life of the radiolabel and the integrity of the antibody is reserved for the therapeutic purpose for which it is labeled.
However, there still remain some impediments to convenient clinical use of immunotherapeutics radiolabeled with beta-emitting radioisotopes such as 90Y. Unlike 111In, 90Y cannot be used for imaging purposes due to the lack of gamma radiation associated therewith. Thus, a diagnostic “imaging” radionuclide, such as 111In, is usually employed for determining the location and relative size of a tumor prior to and/or following administration of therapeutic chimeric or 90Y-labeled antibody. Additionally, indium-labeled antibody enables dosimetric assessment to be made, which is generally believed to be required before 90Y-labeled antibodies are used due to their relatively high potency and tendency to be absorbed in the bones.
For instance, U.S. Pat. No. 5,843,398 of Kaminski et al. discloses a method of administering 90Y-labeled antibodies to a patient having lymphoma, but maintains that dosimetry is required for 90Y. To effect dosimetry, Kaminski uses a 111In-labeled antibody prior to administering the 90Y-labeled antibody, despite acknowledging that some inaccuracy is anticipated due to the different pharmacokinetic characteristics of the radioisotopes. In addition, the Kaminski patent suggests that dose escalation of 90Y-labeled antibodies may also be performed in a cautious progression to minimize the chances of irreversible toxicities.
This requirement for dosimetric evaluation prior to administration of a therapeutic antibody detracts from the convenience of using immunotherapy to treat patients in the clinical setting, wastes precious time during which the patient could be undergoing treatment which will actually help alleviate the disease, and increases the exposure to radioactivity for both the patient and the doctor. Moreover, by using diagnostic antibodies which target the same cell surface molecule as the therapeutic antibody, more time must be allotted for the diagnostic antibodies to clear the system in order for the therapeutic antibodies to have a clear path to their target on the surface of malignant B cells. It would be helpful to the field of immunotherapeutics and further facilitate the use of such therapeutics in the clinical setting if methods for predicting the toxicity of radiolabeled antibodies for each particular patient were developed which would enable the clinician to forgo the need for dosimetry with diagnostic radiolabeled antibodies.